This invention is directed to compositions comprising soluble divalent and multivalent heterodimeric analogs of proteins that are involved in immune regulation and methods of making and using the same. The high affinity that these complexes have for their cognate ligands enables them to be effective competitors to T cell receptors and MHC molecules normally involved in transplant rejection and autoimmune disease. Molecules such as divalent T cell receptors may also have an impact on the diagnosis and treatment of cancer in that they may be used to augment antitumor responses, or may be conjugated to toxins which may then be used to help eliminate tumors. Use of such compositions will allow one to accomplish selective immune modulation without compromising the general performance of the immune system.
The process of signal transduction often involves proteins that have extracellular domains, transmembrane domains, and intracellular domains. During ligand binding there is often oligomerization of receptor molecules in order to transmit effectively the signal to the intracellular component of the cell. The immune system is an excellent example of a signal transduction pathway that works by these methods (Rosen et al. J. Med. Chem. 38: 48-55).
The immune system is a defense system found in most advanced forms of higher vertebrates. A properly functioning lymphatic and immune system distinguishes between self and nonself. A healthy body protects against foreign invaders, such as bacteria, viruses, fungi, and parasites. As the body encounters foreign material (nonself), also known as an antigens, the immune system becomes activated. An antigen is recognized by characteristic shapes or epitopes on its surface. This defense mechanism provides a means of rapid and highly specific responses that are used to protect an organism against invasion by pathogenic microorganisms. It is the myriad of pathogenic microorganisms that have principally caused the evolution of the immune system to its current form. In addition to protection against infectious agents. specific immune responses are thought to be involved in surveillance against alterations in self antigens as seen in tumor development. Immune responses are also involved in the development of autoimmune disease, AIDS, as well as rejection of transplanted tissues.
Lymphocytes
Within the immune system, lymphocytes play a central role. Lymphocyte responses to foreign organisms orchestrate the effector limbs of the immune system, and ultimately, determine the fate of an infection. Lymphocytes can be divided into two main categories, B and T cells. These two types of lymphocytes are specialized in that they have different effector functions and play different roles in the development of specific immune responses. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. Specificity is conferred by an unique set of cell surface receptors expressed on individual lymphocytes. These receptors interact with soluble proteins, in the case of B cells, and with antigenic peptide/major histocompatibility complex (MHC) molecules in the case of T lymphocytes. The nature of the interaction with their ligands also differs between B and T cells. The antigen receptors produced by B cells, immunoglobulins (Igs), interact with their ligands with a high affinity. In contrast, T cell receptors interact with their ligands with low affinity. Thus, the T cell response is driven by the interaction of many T cell receptors (TcR) on the surface of an individual T cell interacting with multiple antigenic peptide/MHC complexes on the surface of the antigen presenting cell. Thus, these two diverse groups of cell-surface glycoproteins, the TcRs and the MHC glycoproteins, form key components of specificity in the T lymphocyte response to antigens.
T cells are a major regulatory cell of the immune system. Their regulatory functions depend not only on expression of a unique T cell receptor, but also on expression of a variety of accessory molecules and effector functions associated with an individual T cell response. Effector functions include responses such as cytotoxic responses or other responses characterized by secretion of effector molecules, i.e., lymphokines. It is this regulatory function that often goes awry in the development of autoimmune diseases. The different effector functions also play a large role in tissue graft rejection, and can be important in tumor rejection.
T cells respond to antigens in the context of either Class I or Class II MHC molecules. Cytotoxic T cells respond mainly against foreign antigens in the context of Class I glycoproteins, such as viral-infected cells, tumor antigens and transplantation antigens. In contrast, helper T cells respond mainly against foreign antigens in the context of Class II molecules. Both types of MHC molecules are structurally distinct, but fold into very similar shapes. Each MHC molecule has a deep groove into which a short peptide, or protein fragment, can bind. Because this peptide is not part of the MHC molecule itself. it varies from one MHC molecule to the next. It is the presence of foreign peptides displayed in the MHC groove that engages clonotypic T cell receptors on individual T cells, causing them to respond to foreign antigens.
Antigen-specific recognition by T cells is based on the ability of clonotypic T cell receptor to discriminate between various antigenic-peptides resident in MHC molecules. These receptors have a dual specificity for both antigen and MHC (Zinkemagel et al. Nature 248: 701-702 (1974)). Thus, T cells are both antigen-specific and MHC-restricted. A simple molecular interpretation of MHC-restricted recognition by T cells is that TcRs recognize MHC residues as well as peptide residues in the MHC-peptide complex. Independent of the exact mechanism of recognition, the clonotypic T cell receptor is the molecule that is both necessary and sufficient to discriminate between the multitude of peptides resident in MHC.
T cells can be divided into two broad subsets; those expressing xcex1/xcex2 TcR and a second set that expresses xcex3/xcex4 TcR. Cells expressing xcex1/xcex2 TcR have been extensively studied and are known to comprise most of the antigen-specific T cells that can recognize antigenic peptide/MHC complexes encountered in viral infections, autoimmune responses, allograft rejection and tumor-specific immune responses. Cells expressing xcex1/xcex2 TcRs can be further divided into cells that express CD8 accessory molecules and cells that express CD4 accessory molecules. While there is no intrinsic difference between the clonotypic xcex1/xcex2 T cell receptors expressed either on CD4 and CD8 positive cells, the accessory molecules largely correlate with the ability of T cells to respond to different classes of MHC molecules. Class I MHC molecules are recognized by CD8+, or cytotoxic, T cells and class II MHC molecules by CD4+, or helper, T cells.
xcex3/xcex4 T cells make up another significant population of T cells seen in circulation as well as in specific tissues. These cells are not well understood; their antigen/MHC specificity is poorly defined and in most cases their ligands arc completely unknown. These cells are present in high quantities in certain tissues, including skin and gut epithelium, and are thought to play a significant role in immune responses of those organs. They have also been implicated in autoimmune responses and may be involved in the recognition of heat shock proteins. A general approach to the identification of antigenic complexes, as outlined in the present invention, would greatly facilitate understanding of how these cells influence the development of both normal and abnormal immune responses. There is a large degree of homology between both xcex1/xcex2 and xcex3/xcex4 TcR expressed in rodents and humans. This extensive homology has, in general, permitted one to develop murine experimental models from which results and implications may be extrapolated to the relevant human counterpart.
MHC Molecules in Health and Disease
Major histocompatibility antigens consist of a family of antigens encoded by a complex of genes called the major histocompatibility complex. In mice, MHC antigens are called H-2 antigens (Histocompatibility-2 antigens). In humans MHC antigens are called HLA antigens (Human-Leukocyte-associated Antigens). The loci that code for MHC glycoproteins are polymorphic. This means that each species has several different alleles at each locus. For example, although a large number of different Class I antigens may be seen in a species as a whole, any individual inherits only a single allele from each parent at each locus. and therefore expresses at most two different forms of each Class I antigen.
In the murine system, the class II MHC molecules are encoded by I-A and I-E loci, and in humans, class II molecules are encoded by the DR, DP and DQ loci. Polymorphism of class II alleles is attributed to the alpha and beta chains and specificities are designated using the nomenclature set forth by the World Health Organization (Immunogenetics (1992) 36:135).
The Role of MHC Molecules-Transplantation
MHC molecules play an essential role in determining the fate of grafts. Various species display major immunological functional properties associated with the MHC including, but not limited to, vigorous rejection of tissue grafts, stimulation of antibody production, stimulation of the mixed lymphocyte reaction (MLR), graft-versus-host reactions (GVH), cell-mediated lympholysis (CML), immune response genes, and restriction of immune responses. Transplant rejection occurs when skin, organs (e.g., kidney, liver, lung), or other tissues (e.g., blood, bone marrow) are transplanted across an MHC incompatibility. A vigorous graft rejection occurs when the immune system is activated by mismatched transplantation antigens that are present in donor tissue but not in recipient. Graft rejection may occur in the graft itself by exposure of circulating immune cells to foreign antigens, or it may occur in draining lymph nodes due to the accumulation of trapped transplantation antigens or graft cells. Because of the extensive diversity of MHC antigens, numerous specificities are possible during physiological and pathophysiologic immune-related activities, (e.g., transplantation, viral infections, and tumor development). The recognized HLA specificities are depicted, for example, in a review by Bodmer et al. (In: Dupont B. (Ed.) Immunobiology of HLA (Volume I) New York: Springer-Verlag (1989)).
The Role of MHC Molecules-Autoimmune Response
Susceptibility to many autoimmune disease shows a significant genetic component and familial linkage. Most genetic linkages of autoimmune diseases are with certain class II MHC alleles (see Table 1 for Overview). The level of association between a particular disease and an allele at one of the MHC loci is defined by a term called xe2x80x9crelative riskxe2x80x9d. This term reflects the frequency of the disease in individuals who have the antigen compared to the frequency of the disease among individuals who lack the antigens. For example, there is a strong association with DQxcex2 genotype in insulin-dependent diabetes mellitus; the normal DQxcex2 sequence has an aspartic acid at position 57, whereas in Caucasoid populations, patients with diabetes most often have valine, serine or alanine at that position.
Regulation of Immune Reponses
Interest in analyzing both normal and abnormal T cell-mediated immune responses led to the development of a series of novel soluble analogs of T cell receptors and MHC molecules to probe and regulate specific T cell responses. The development of these reagents was complicated by several facts. First, T cell receptors interact with peptide/MHC complexes with relatively low affinities (Matsui et al Science 254:1788-1891 (1991) Sykulev et al Immunity 1:15-22 (1994) Corr et al Science 265:946-949 (1994)). In order to specifically regulate immune responses, soluble molecules with high affinities/avidities for either T cell receptors or peptide/MHC complexes are needed. However, simply making soluble monovalent analogs of either T cell receptors or peptide/MHC complexes has not proven to be effective at regulating immune responses with the required specificity and avidity.
To regulate immune responses selectively, investigators have made soluble versions of proteins involved in immune responses. Soluble divalent analogs of proteins involved in regulating immune responses with single transmembrane domains have been generated by several laboratories. Initially, CD4/Ig chimeras were generated (Capon et al Nature 337:525-531 (1989); Bryn et al Nature 344:667-670 (1990)), as well as CR2/Ig chimeras (Hebell et al Science 254:102-105 (1991)). Later it was demonstrated that immune responses could be modified using specific CTLA-4/Ig chimeras (Linsley et al Science 257:7920-795 (1992); U.S. Pat. No. 5,434,131; Lenschow et al Science 257:789-791 (1992)). In addition, class I MHC/Ig chimeras were used to modify in vitro allogeneic responses (Dal Porto, supra). However, these examples include only soluble divalent analogs of single transmembrane polypeptide molecules and not chimeric molecules of heterodimeric proteins in which the heterodimer consists of xcex1 and xcex2 polypeptides that are both transmembrane polypeptides. The present invention reports the generation of soluble divalent and multivalent heterodimeric analogs of integral membrane protein complexes, which consist of alpha and beta polymorphic integral membrane polypeptides that properly fold to form a functional unit that has potential use in immune modulation.
Previously, replacement of two transmembrane domains in the generation of multivalent analogs has not been achieved. The challenge of generating these molecules lies in achieving the proper folding and expression of two polypeptides, both of which ordinarily require transmembrane domains (FIG. 1). In addition, soluble multivalent analogs of heterodimeric proteins generally have increased affinity and, therefore, are preferred therapeutic agents. These soluble protein complexes, which consist of xcex1 and xcex2 polymorphic integral membrane polypeptides that properly fold to form a functional unit, have potential use as immune modulating agents.
It is one objective of this invention to provide soluble recombinant divalent and multivalent analogs of heterodimeric proteins, which are capable of specifically binding target molecules to regulate immune responses.
It is another object of this invention to provide soluble recombinant divalent heterodimeric proteins that possess enhanced affinity for their target molecules.
It is still another object of this invention to claim a method for producing an expression vector encoding soluble divalent analogs of heterodimeric integral membrane proteins. This comprises modifying an expression vector for an immunoglobulin molecule by inserting at least two DNA sequences, such as an a polypeptide fused to an immunoglobulin heavy chain and a xcex2 polypeptide fused to an immunoglobulin light chain (FIG. 2). This could also be done by inserting at least two DNA sequences, such as a xcex2 polypeptide fused to an immunoglobulin heavy chain and an xcex1 polypeptide fused to an immunoglobulin light chain. The xcex1 and xcex2 polypeptides are ones that encode a binding or recognition site. It is also possible for the fusion proteins of the present invention to be encoded by two compatible expression vectors.
A host cell containing the vector or vectors, which is capable of expressing soluble divalent heterodimeric proteins containing an xcex1 and xcex2 polypeptide subunit is also an object of this invention.
Also included in this invention is a method for inhibiting or decreasing immune responses. Specifically, antigen-specific interactions between T cells and cells presenting antigens may be inhibited using the soluble divalent analogs of either the TcR or class II MHC molecules. An example of this could be suppression of an autoimmune response as seen in Myasthenia gravis, multiple sclerosis, arthritis, and allergic diseases. Adhesion of cells mediated through the interactions of integrins can also be inhibited using soluble divalent analogs of integrin molecules. Inhibition of cytokine-mediated cell stimulation is also included, in that soluble divalent versions of cytokine receptors could bind to soluble cytokines, thereby inhibiting the ability of the cytokines to mediate cellular proliferation.
Also included in this invention is a method for augmenting immune responses. Specifically, antigen-specific interactions between T cells and cells presenting antigens may be augmented using the soluble divalent analogs of either the TcR or class II MHC molecules immobilized on a substrate to stimulate antigen-specific T cell responses. Such a system may also be used, in the case of immobilized MHC/Ig molecules presenting antigenic peptides, to identify and purify specific T cell subsets, i.e. for the identification of the clonotypic TcR. Along the same lines, immobilized TcR/Ig may be used to identify and purify unknown peptide/MHC complexes which may be involved in cancer or infectious diseases such as AIDS. Stimulation of cells via adhesion receptors can also be accomplished using soluble divalent analogs of integrin molecules that have been immobilized on a solid substrate, such as a tissue culture plate or bead.
The invention further includes a method for treating diseases by administering soluble recombinant divalent heterodimeric analogs of proteins whereby the xcex1 and xcex2 polypeptides form a unit, and whereby the claimed constructs selectively increase or decrease cellular activation, proliferation, anergy, or deletion of specific T cell subsets. Such diseases include autoimmune disorders, transplant rejection, cancer and AIDS. Since divalent and multivalent complexes of the present invention will have increased affinity for their respective targets, administration of such compounds should selectively suppress or block T cell recognition of specific transplantation antigens and self antigens by binding to the designated target molecule and inhibiting cell-to-cell interaction.
It is also possible using techniques known in the art to conjugate toxin molecules, such as ricin and pseudomonas exotoxin, to the compounds of the present invention. The invention also includes methods of treating cancer and AIDS with such conjugate molecules. For example, following the identification of virus- or tumor-specific peptides displayed on the MHC molecules of viral-infected or tumor cells, toxin-conjugated soluble heterodimeric TCR molecules may be designed that bind to and destroy cells harboring the HIV virus, or cancerous cells, respectively. In addition, soluble divalent or multivalent MHC/IG molecules displaying tumor- or AIDS-related peptides might have potential use in immunization protocols.
Accordingly, also included in the invention are methods of identifying unknown antigens or peptides derived using soluble divalent TcR. A distinct advantage of soluble high affinity TCR/Ig chimeras is that even in the absence of any a priori knowledge about their ligands, they may be useful in defining the specific peptide/MHC ligands recognized by uncharacterized tumor-specific T cells and T cells involved in autoimmune responses. Not only are soluble divalent TCR/IG molecules efficient probes for the quantitative detection of specific peptide/MHC complexes, but due to their strong affinity for the target molecule, they will consequently play an important role in the purification of such complexes and facilitate their characterization.
Soluble divalent heterodimeric analogs of integral membrane proteins of this invention provide significant benefits because these recombinant proteins possess enhanced binding affinities for modulating immune responses. High affinity divalent ligands, such as the divalent chimeric molecules of this invention, can be used to selectively modulate specific r cell responses and to study cell-cell interactions that are driven by multivalent ligand-receptor interactions.